Use of interleukin-19 to treat ovarian cancer

ABSTRACT

The present invention relates to the anti-cancer activity of IL-19 polypeptide molecules. IL-19 is a cytokine involved in inflammatory processes and human disease. The present invention includes Use of IL-19 for decreasing proliferation of ovarian cancer cells, treating ovarian cancer, amongst other uses disclosed. IL-19 polypeptides can be administered alone, or can be fused to cytotoxic moieties, and can be administered in conjunction with radiation or chemotherapeutic agents.

REFERENCE TO RELATED APPLICATIONS

[0001] The present application is related to U.S. ProvisionalApplication Serial No. 60/372,344 filed Apr. 11, 2002. Under 35 U.S.C.§119(e)(1), this application claims benefit of said ProvisionalApplications.

BACKGROUND OF THE INVENTION

[0002] Cancer of the ovary is the leading cause of death fromgynecologic malignancies and the fourth common cause of cancer-relateddeath among women. This is in spite of the fact that the occurrence ofovarian cancer is relatively rare. Only 1.5% of women develop thedisease, and it is only the seventh most common cause of cancer inwomen.

[0003] Ovarian cancer can be divided into three sub-types depending onthe cell type involved, namely, epithelial, stromal and germ celltumors.

[0004] At least 80% of malignant ovarian tumors arise form the coelomicepithelium. The most common type is serous crystadenocarcinoma, whichaccounts for 75% of cases of epithelial ovarian cancer.

[0005] Most women (75%) present with advanced-stage disease, and mosthave vague, nonspecific symptoms, such as dyspepsia, bloating,early-satiety anorexia, gas pains and backache. The most common earlyfinding is an adnexal mass, which is often solid, irregular, and fixed.A patient may be asymptomatic until the disease is advanced.Occasionally, a patient presents with severe abdominal pain secondary totorsion of the ovarian mass. Late in the course, pelvic pain, anemia,cachexia, and abdominal swelling due to ovarian enlargement oraccumulation of ascitic fluid usually occur. Nodular implants noted onthe rectovaginal examination suggest extensive pelvic malignant disease.

[0006] Stromal tumors constitute only a tenth of ovarian malignanciesbut account for most of the hormone-secreting tumors.

[0007] Germ cell tumors comprise less than 5 percent of ovarianmalignancies, occur in young women, and have a higher incidence inAfrican-American women than Caucasian women. Functional effects of germcell or stromal tumors include hyperthyroidism, feminization, andvirilization.

[0008] After surgery to remove the tumor chemotherapy is usuallyprovided. The initial chemotherapeutic regimen is three to six coursesof chemotherapy. Paclitaxel is combined with cisplatin or carboplatin.Other chemotherapeutic drugs include topotecan, hexamethylmelamine,ifosfamide, doxorubicin, bleomycin and etoposide. In spite of theregimens, the five-year survival rate of patients with stage II diseaseis only fifty to seventy percent and thirty to forty percent forpatients with stage III disease.

[0009] Thus, there is a need for new therapeutics that can be used totreat ovarian cancer.

DESCRIPTION OF THE INVENTION

[0010] The present invention fills this need by administeringinterleukin-19 (IL-19) to a mammalian having ovarian cancer. The presentinvention also provides a method for inhibiting the growth of ovariancancer cells by bringing IL-19 or fragments comprising helices A-D ofIL-19, into contact with said cancerous ovarian cells. Interleukin-19,and fragments comprising helices A-D of IL-19, can be produced accordingto the method described in U.S. Pat. No. 5,985,614. The polynucleotidesequence of IL-19 is shown in SEQ ID NO:1 and corresponding amino acidsequence is shown in SEQ ID NO:2; the mature secreted form of the IL-19polypeptide is shown from amino acid number 23 (His) to 177 (Ala) of SEQID NO:2.

[0011] The quantities of IL-19 for effective therapy will depend uponmany different factors, including means of administration, target site,physiological state of the patient, and other medications administered.Thus, treatment dosages should be titrated to optimize safety andefficacy. Typically, dosages used in vitro may provide useful guidancein the amounts useful for in vivo administration of these reagents.Animal testing of effective doses for treatment of particular disorderswill provide further predictive indication of human dosage. Methods foradministration include, intravenous, peritoneal, intramuscular,transdermal or administration into the lung or trachea in spray form bymeans or a nebulizer or atomizer. Pharmaceutically acceptable carrierswill include water, saline, buffers to name just a few. Dosage rangeswould ordinarily be expected from 1 μg to 1000 μg per kilogram of bodyweight per day. However, the doses may be higher or lower as can bedetermined by a medical doctor with ordinary skill in the art.Excipients and stabilizers can possible be added. These include glycine,histidine, glutamate, aspartate, sugars, sucrose, trehalose, galactosesorbitol, arginine, D-and/or L amino acids, sugar alcohols, lactose,maltose, threonine, lysine, methionine, isoleucine, a surface activeagent such as TWEEN 80, TWEEN 20, polyethylene glycol (PEG)(particularly those PEGs having molecular weights between 1000 and 35000Da), cetyl alcohol, polyvinylpyrrolidone, polyvinyl alcohol, lanolinalcohol and sorbitan. A reducing agent may be included, such ascysteine, N-acetyl-cysteine, and thioglycerol. For a complete discussionof drug formulations and dosage ranges see Remington's PharmaceuticalSciences, 18^(th) Ed., (Mack Publishing Co., Easton, Pa., 1996), andGoodman and Gilman's: The Pharmacological Bases of Therapeutics, 9^(th)Ed. (Pergamon Press 1996).

[0012] In addition, as IL-19 is useful in treating ovarian orcervical-specific cancers, the anti-tumor and anti-proliferativeactivity and effect of IL-19 on tumor progression and metastasis can bemeasured in vivo. Several syngeneic mouse models have been developed tostudy the influence of polypeptides, compounds or other treatments ontumor progression. In these models, tumor cells passaged in culture areimplanted into mice of the same strain as the tumor donor. The cellswill develop into tumors having similar characteristics in the recipientmice, and metastasis will also occur in some of the models. Appropriatetumor models for our studies include the Lewis lung carcinoma (ATCC No.CRL-1642) and B16 melanoma (ATCC No. CRL-6323), amongst others. Theseare both commonly used tumor lines, syngeneic to the C57BL6 mouse, thatare readily cultured and manipulated in vitro. Tumors resulting fromimplantation of either of these-cell lines are capable of metastasis tothe lung in C57BL6 mice. The Lewis lung carcinoma model has recentlybeen used in mice to identify an inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79: 315-328,1994). C57BL6/J mice are treated with anexperimental agent either through daily injection of recombinantprotein, agonist or antagonist or a one-time injection of recombinantadenovirus. Three days following this treatment, 10⁵ to 10⁶ cells areimplanted under the dorsal skin. Alternatively, the cells themselves maybe infected with recombinant adenovirus, such as one expressing IL-19,before implantation so that the protein is synthesized at the tumor siteor intracellularly, rather than systemically. The mice normally developvisible tumors within 5 days. The tumors are allowed to grow for aperiod of up to 3 weeks, during which time they may reach a size of1500-1800 mm³ in the control treated group. Tumor size and body weightare carefully monitored throughout the experiment. At the time ofsacrifice, the tumor is removed and weighed along with the lungs and theliver. The lung weight has been shown to correlate well with metastatictumor burden. As an additional measure, lung surface metastases arecounted. The resected tumor, lungs and liver are prepared forhistopathological examination, immunohistochemistry, and in situhybridization, using methods known in the art and described herein. Theinfluence of the expressed polypeptide in question, e.g., IL-19, on theability of the tumor to recruit vasculature and undergo metastasis canthus be assessed. In addition, aside from using adenovirus, theimplanted cells can be transiently transfected with IL-19. Use of stableIL-19 transfectants as well as use of induceable promoters to activateIL-19 expression in vivo are known in the art and can be used in thissystem to assess IL-19 induction of metastasis. Moreover, purified IL-19or IL-19-conditioned media can be directly injected in to this mousemodel, and hence be used in this system. For general reference see,O'Reilly M S, et al. Cell 79:315-328, 1994; and Rusciano D, et al.Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.

[0013] Similarly, animal tumor models such as human xenograft models inimmunocompromised animals are used for cervical and ovarian cancermodels and are known in the art. For example, one ovarian carcinomamodel is as follows: NIH:OVCAR-5 cells injected into Swiss nude mice, asdisclosed in Molpus, K L et al, Int. J. Cancer 68:588-95 (1996), whichcharacterizes a xenograft model of human ovarian carcinoma whichproduces intraperitoneal carcinomatosis and metastases in mice. Forexample, one cervical carcinoma model is as follows: Cervical carcinoma:ME180 and SiHa human cervical squamous cell carcinoma lines grown inSCID mice. See, Moreno-Merlo F et al, Br. J. Cancer 81: 989-93 (1999)and Vukovic, V. et al, Int. J. Radiat Oncol Biol Phys 52:837-43 (2002).

[0014] Suitable detectable molecules may be directly or indirectlyattached to the IL-19 polypeptide, and include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like. Suitable cytotoxic moleculesmay be directly or indirectly attached to the polypeptide, and includebacterial or plant toxins (for instance, diphtheria, toxin, saporin,Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeuticradionuclides, such as iodine-131, rhenium-188 or yttrium-90 (eitherdirectly attached to the polypeptide, or indirectly attached throughmeans of a chelating moiety, for instance). Polypeptides may also beconjugated to cytotoxic drugs, such as adriamycin. For indirectattachment of a detectable or cytotoxic molecule, the detectable orcytotoxic molecule can be conjugated with a member of acomplementary/anticomplementary pair, where the other member is bound tothe polypeptide. For these purposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

[0015] In addition, IL-19 polypeptide-toxin fusion proteins can be usedfor targeted cell or tissue inhibition or ablation (for instance, totreat cancer cells or tissues). Alternatively, if the polypeptide hasmultiple functional domains (i.e., an activation domain or a receptorbinding domain, plus a targeting domain), a fusion protein includingonly the targeting domain may be suitable for directing a cytokine(e.g., IL-19), a detectable molecule, a cytotoxic molecule or acomplementary molecule to a cell or tissue type of interest, e.g., toovarian or cervical tissue. In instances where the domain only fusionprotein includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting carrier or vehicle for cell/tissue-specific delivery ofgeneric anti-complementary-detectable/cytotoxic molecule conjugates.

[0016] In another embodiment, IL-19 cytokine fusion proteins can be usedfor in vivo killing of target tissues (for example, ovarian cancer, orcervical cancer, or leukemia, lymphoma, lung cancer, colon cancer,melanoma, pancreatic cancer, skin, blood and bone marrow cancers, orother cancers wherein IL-19 receptors are expressed) (See, generally,Chang, C. H. et al, Mol Cancer Ther 7:553-63(2002)). The describedfusion proteins enable targeting of a cytokine to a desired site ofaction, thereby providing an elevated local concentration of cytokine.Suitable IL-19 polypeptides target an undesirable cell or tissue (i.e.,a tumor or a leukemia), and the fused cytokine mediated improved targetcell lysis by effector cells. Suitable cytokines for this purposeinclude interleukin 2 and granulocyte-macrophage colony-stimulatingfactor (GM-CSF), for instance.

[0017] In yet another embodiment, if the IL-19 polypeptide targets tumorcells or cancerous tissues, such polypeptide may be conjugated with aradionuclide, and particularly with a beta-emitting radionuclide, toreduce restenosis (e.g., in vascular tissue). Such therapeuticapproaches pose less danger to clinicians who administer the radioactivetherapy. For instance, iridium-192 impregnated ribbons placed intostented vessels of patients until the required radiation dose wasdelivered showed decreased tissue growth in the vessel and greaterluminal diameter than the control group, which received placebo ribbons.Further, revascularisation and stent thrombosis were significantly lowerin the treatment group. Similar results are predicted with targeting ofa bioactive conjugate containing a radionuclide, as described herein.

[0018] The bioactive polypeptide described herein can be deliveredintravenously, intraarterially or intraductally, or may be introducedlocally at the intended site of action.

[0019] For pharmaceutical use, the IL-19 are formulated for parenteral,particularly intravenous or subcutaneous, delivery according toconventional methods. Intravenous administration will be by bolusinjection, controlled release, e.g, using mini-pumps or otherappropriate technology, or by infusion over a typical period of one toseveral hours. In general, pharmaceutical formulations will include aprotein in combination with a pharmaceutically acceptable vehicle, suchas saline, buffered saline, 5% dextrose in water or the like.Formulations may further include one or more excipients, preservatives,solubilizers, buffering agents, albumin to provent protein loss on vialsurfaces, etc. In addition, the IL-19 may be combined with othercytokines, particularly early-acting cytokines such as stem cell factor,IL-3, IL-6, IL-11 or GM-CSF. When utilizing such a combination therapy;the cytokines may be combined in a single formulation or may beadministered in separate formulations. Methods of formulation are wellknown in the art and are disclosed, for example, in Remington'sPharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton Pa.,1990, which is incorporated herein by reference. Therapeutic doses willgenerally be in the range of 0.1 to 100 mg/kg of patient weight per day,preferably 0.5-20 mg/kg per day, with the exact dose determined by theclinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.The proteins will commonly be administered over a period of up to 28days following chemotherapy or bone-marrow transplant or until aplatelet count of >20,000/mm³, preferably >50,000/mm³, is achieved. Morecommonly, the proteins will be administered over one week or less, oftenover a period of one to three days. In general, a therapeuticallyeffective amount of IL-19 is an amount sufficient to produce aclinically significant increase in the proliferation and/ordifferentiation of lymphoid or myeloid progenitor cells, which will bemanifested as an increase in circulating levels of mature cells (e.g.platelets or neutrophils). Treatment of platelet disorders will thus becontinued until a platelet count of at least 20,000/mm³, preferably50,000/mm³, is reached. The IL-19 can also be administered incombination with other cytokines such as IL-3, -6 and -11; stem cellfactor; erythropoietin; G-CSF and GM-CSF. Within regimens of combinationtherapy, daily doses of other cytokines will in general be: EPO, 150U/kg; GM-CSF, 5-15 lg/kg; IL-3, 1-5 lg/kg; and G-CSF, 1-25 lg/kg.Combination therapy with EPO, for example, is indicated in anemicpatients with low EPO levels.

[0020] For pharmaceutical use, the IL-19 polypeptides of the presentinvention are formulated for parenteral, particularly intravenous orsubcutaneous, delivery according to conventional methods. Intravenousadministration will be by bolus injection or infusion over a typicalperiod of one to several hours. In general, pharmaceutical formulationswill include a IL-19 protein in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. Methods of formulation arewell known in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Therapeutic doses will generally be in therange of 0.1 to 100 μg/kg of patient weight per day, preferably 0.5-20mg/kg per day, with the exact dose determined by the clinician accordingto accepted standards, taking into account the nature and severity ofthe condition to be treated, patient traits, etc. Determination of doseis within the level of ordinary skill in the art. The proteins may beadministered for acute treatment, over one week or less, often over aperiod of one to three days or may be used in chronic treatment, overseveral months or years. In general, a therapeutically effective amountof IL-19 is an amount sufficient to produce a clinically significantchange in a cancer, cell growth or immune function.

[0021] The present invention also contemplates chemically modified IL-19polypeptide is linked with a polymer. Illustrative IL-19 polypeptidesare soluble polypeptides comprising a mature IL-19 polypeptide or afragment of the IL-19 polypeptide comprising helices A-D of thepolypeptide. Typically, the polymer is water soluble so that the IL-19polypeptide conjugate does not precipitate in an aqueous environment,such as a physiological environment. An example of a suitable polymer isone that has been modified to have a single reactive group, such as anactive ester for acylation, or an aldehyde for alkylation, In this way,the degree of polymerization can be controlled. An example of a reactivealdehyde is polyethylene glycol propionaldehyde, or mono-(C1-C10)alkoxy, or aryloxy derivatives thereof (see, for example, Harris, etal., U.S. Pat. No. 5,252,714). The polymer may be branched orunbranched. Moreover, a mixture of polymers can be used to produce IL-19polypeptide conjugates.

[0022] IL-19 polypeptide conjugates used for therapy can comprisepharmaceutically acceptable water-soluble polymer moieties. Suitablewater-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde,bis-succinimidyl carbonate PEG, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or othercarbohydrate-based polymers. Suitable PEG may have a molecular weightfrom about 600 to about 60,000, including, for example, 5,000, 12,000,20,000 and 25,000. An IL-19 polypeptide conjugate can also comprise amixture of such water-soluble polymers.

[0023] One example of a IL-19 polypeptide conjugate comprises an IL-19polypeptide moiety and a polyalkyl oxide moiety attached to theN-terminus of the IL-19 polypeptide moiety. PEG is one suitablepolyalkyl oxide. As an illustration, IL-19 polypeptide can be modifiedwith PEG, a process known as “PEGylation.” PEGylation of IL-19polypeptide can be carried out by any of the PEGylation reactions knownin the art (see, for example, EP 0 154 316, Delgado et al., CriticalReviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan andSpreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int JHematol 68:1 (1998)). For example, PEGylation can be performed by anacylation reaction or by an alkylation reaction with a reactivepolyethylene glycol molecule. In an alternative approach, IL-19polypeptide conjugates are formed by condensing activated PEG, in whicha terminal hydroxy or amino group of PEG has been replaced by anactivated linker (see, for example, Karasiewicz et al., U.S. Pat. No.5,382,657).

[0024] PEGylation by acylation typically requires reacting an activeester derivative of PEG with an IL-19 polypeptide. An example of anactivated PEG ester is PEG esterified to N-hydroxysuccinimide. As usedherein, the term “acylation” includes the following types of linkagesbetween IL-19 polypeptide and a water soluble polymer: amide, carbamate,urethane, and the like. Methods for preparing PEGylated IL-19polypeptide by acylation will typically comprise the steps of (a)reacting a IL-19 polypeptide with PEG (such as a reactive ester of analdehyde derivative of PEG) under conditions whereby one or more PEGgroups attach to 1L-19 polypeptide, and (b) obtaining the reactionproduct(s). Generally, the optimal reaction conditions for acylationreactions will be determined based upon known parameters and desiredresults. For example, the larger the ratio of PEG: IL-19 polypeptide,the greater the percentage of polyPEGylated IL-19 polypeptide product.

[0025] The product of PEGylation by acylation is typically apolyPEGylated IL-19 polypeptide product, wherein the lysine ε-aminogroups are PEGylated via an acyl linking group. An example of aconnecting linkage is an amide. Typically, the resulting IL-19polypeptide will be at least 95% mono-, di-, or tri-pegylated, althoughsome species with higher degrees of PEGylation may be formed dependingupon the reaction conditions. PEGylated species can be separated fromunconjugated IL-19 polypeptides using standard purification methods,such as dialysis, ultrafiltration, ion exchange chromatography, affinitychromatography, and the like.

[0026] PEGylation by alkylation generally involves reacting a terminalaldehyde derivative of PEG with IL-19 polypeptide in the presence of areducing agent. PEG groups can be attached to the polypeptide via a—CH₂—NH group.

[0027] Derivatization via reductive alkylation to produce amonoPEGylated product takes advantage of the differential reactivity ofdifferent types of primary amino groups available for derivatization.Typically, the reaction is performed at a pH that allows one to takeadvantage of the pKa differences between the ε-amino groups of thelysine residues and the α-amino group of the N-terminal residue of theprotein. By such selective derivatization, attachment of a water-solublepolymer that contains a reactive group such as an aldehyde, to a proteinis controlled. The conjugation with the polymer occurs predominantly atthe N-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups. The presentinvention provides a substantially homogenous preparation of IL-19polypeptide monopolymer conjugates.

[0028] Reductive alkylation to produce a substantially homogenouspopulation of monopolymer IL-19 polypeptide conjugate molecule cancomprise the steps of: (a) reacting a IL-19 polypeptide with a reactivePEG under reductive alkylation conditions at a pH suitable to permitselective modification of the α-amino group at the amino terminus of theIL-19 polypeptide, and (b) obtaining the reaction product(s). Thereducing agent used for reductive alkylation should be stable in aqueoussolution and able to reduce only the Schiff base formed in the initialprocess of reductive alkylation. Illustrative reducing agents includesodium borohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

[0029] For a substantially homogenous population of monopolymer IL-19polypeptide conjugates, the reductive alkylation reaction conditions arethose that permit the selective attachment of the water-soluble polymermoiety to the N-terminus of IL-19 polypeptide. Such reaction conditionsgenerally provide for pKa differences between the lysine amino groupsand the α-amino group at the N-terminus. The pH also affects the ratioof polymer to protein to be used. In general, if the pH is lower, alarger excess of polymer to protein will be desired because the lessreactive the N-terminal α-group, the more polymer is needed to achieveoptimal conditions. If the pH is higher, the polymer: IL-19 polypeptideneed not be as large because more reactive groups are available.Typically, the pH will fall within the range of 3 to 9, or 3 to 6.

[0030] Another factor to consider is the molecular weight of thewater-soluble polymer. Generally, the higher the molecular weight of thepolymer, the fewer number of polymer molecules which may be attached tothe protein. For PEGylation reactions, the typical molecular weight isabout 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa, or about 12kDa to about 25 kDa. The molar ratio of water-soluble polymer to IL-19polypeptide will generally be in the range of 1:1 to 100:1. Typically,the molar ratio of water-soluble polymer to IL-19 polypeptide will be1:1 to 20:1 for polyPEGylation, and 1:1 to 5:1 for monoPEGylation.

[0031] General methods for producing conjugates comprising a polypeptideand water-soluble polymer moieties are known in the art. See, forexample, Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al.,U.S. Pat. No. 5,738,846, Nieforth et al., Clin. Pharnacol. Ther. 59:636(1996), Monkarsh et al., Anal. Biochem. 247:434 (1997)). This method canbe employed for making IL-19 polypeptide-comprising homodimeric,heterodimeric or multimeric soluble receptor conjugates.

[0032] A pharmaceutical composition comprising IL-19 polypeptides can befurnished in liquid form, in an aerosol, or in solid form. Liquid forms,are illustrated by injectable solutions, aerosols, droplets, topologicalsolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms. The latter form is illustrated byminiosmotic pumps and implants (Bremer et al., Pharm. Biotechnol. 10:239(1997); Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems,Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer etal., “Protein Delivery with Infusion Pumps,” in Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 239-254 (PlenumPress 1997); Yewey et al., “Delivery of Proteins from a ControlledRelease Injectable Implant,” in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)). Othersolid forms include creams, pastes, other topological applications, andthe like.

[0033] Liposomes provide one means to deliver therapeutic polypeptidesto a subject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

[0034] Liposomes can adsorb to virtually any type of cell and thenslowly release the encapsulated agent. Alternatively, an absorbedliposome may be endocytosed by cells that are phagocytic. Endocytosis isfollowed by intralysosomal degradation of liposomal lipids and releaseof the encapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci.446:368 (1985)). After intravenous administration, small liposomes (0.1to 1.0 μm) are typically taken up by cells of the reticuloendothelialsystem, located principally in the liver and spleen, whereas liposomeslarger than 3.0 μm are deposited in the lung. This preferential uptakeof smaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

[0035] The reticuloendothelial system can be circumvented by severalmethods including saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

[0036] Liposomes can also be prepared to target particular cells ororgans by varying phospholipid composition or by inserting receptors orligands into the liposomes. For example, liposomes, prepared with a highcontent of a nonionic surfactant, have been used to target the liver(Hayakawa et al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm.Bull. 16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

[0037] Alternatively, various targeting ligands can be bound to thesurface of the liposome, such as antibodies, antibody fragments,carbohydrates, vitamins, and transport proteins. For example, liposomescan be modified with branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

[0038] In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

[0039] IL-19 polypeptides with IL-19 receptor binding activity can beencapsulated within liposomes using standard techniques of proteinmicroencapsulation (see, for example, Anderson et al., Infect. Immun.31:1099 (1981), Anderson et al., Cancer Res. 50:1853 (1990), and Cohenet al., Biochim. Biophys. Acta 1063:95 (1991), Alving et al.“Preparation and Use of Liposomes in Immunological Studies,” in LiposomeTechnology, 2nd Edition, Vol. III, Gregoriadis (ed.), page 317 (CRCPress 1993), Wassef et al., Meth. Enzymol. 149:124 (1987)). As notedabove, therapeutically useful liposomes may contain a variety ofcomponents. For example, liposomes may comprise lipid derivatives ofpoly(ethylene glycol) (Allen et al., Biochim. Biophys. Acta 1150:9(1993)).

[0040] Degradable polymer microspheres have been designed to maintainhigh systemic levels of therapeutic proteins. Microspheres are preparedfrom degradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

[0041] The present invention also contemplates chemically modified IL-19polypeptides, for example IL-19 polypeptides linked with a polymer, asdiscussed above.

[0042] Other dosage forms can be devised by those skilled in the art, asshown, for example, by Ansel and Popovich, Pharmaceutical Dosage Formsand Drug Delivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro(ed.), Remington's Pharmaceutical Sciences, 19^(th) Edition (MackPublishing Company 1995), and by Ranade and Hollinger, Drug DeliverySystems (CRC Press 1996).

[0043] The present invention contemplates compositions comprising apeptide or polypeptide described herein. Such compositions can furthercomprise a carrier. The carrier can be a conventional organic orinorganic carrier. Examples of carriers include water, buffer solution,alcohol, propylene glycol, macrogol, sesame oil, corn oil, and the like.

[0044] IL-19 can also me administered in conjunction with othertreatments for ovarian cancer such as surgery and chemotherapy. Examplesof chemotherapeutic agents include but are not limited to paclitaxel,cisplatin, carboplatin, topotecan, hexamethylmelamine, ifosfamide,doxorubicin, bleomycin, Taxol, and etoposide.

[0045] Within one aspect, the present invention provides a method forinhibiting the growth and or proliferation of ovarian cancer cellscomprising bringing IL-19 polypeptide into contact with the ovariancancer cells in an amount sufficient to inhibit or reduce theproliferation of the ovarian cancer cells.

[0046] Within a second aspect, the present invention provides a methodfor treating a female mammal afflicted with ovarian cancer comprisingadministering to the female an isolated IL-19 polypeptide an amount of acomposition of IL-19 polypeptide sufficient to inhibit or reduce theproliferation of the ovarian cancer. In one embodiment, the method is asdescribed above, wherein the IL-19 polypeptide is administered inconjunction with radiation. In another embodiment, the method is asdescribed above, wherein the IL-19 polypeptide is administered inconjunction with a chemotherapeutic agent. In another embodiment, themethod is as described above, wherein the chemotherapeutic agent isselected from the group consisting of paclitaxel, cisplatin,carboplatin, topotecan, hexamethylmelamine, ifosfamide, doxorubicin,bleomycin, Taxol, and etoposide.

[0047] Within a third aspect, the present invention provides a methodfor treating a female mammal afflicted with ovarian cancer comprisingadministering to the female an isolated IL-19 polypeptide an amount of acomposition of IL-19 polypeptide sufficient to inhibit or reduce theproliferation of the ovarian cancer, and wherein the IL-19 polypeptideis fused with a cytotoxic moiety. In another embodiment, the method isas described above, wherein the cytotoxic moiety is a bacterial or planttoxin, cytotoxic radionuclide or cytotoxic drug.

[0048] Within another aspect, the present invention provides a method ofreducing proliferation of ovarian cancer cells comprising administeringto a mammal with a ovarian neoplasm an amount of a composition of IL-19polypeptide sufficient to reduce proliferation of the neoplastic ovariancells. In one embodiment, the method is as described above, wherein theIL-19 polypeptide is administered in conjunction with radiation. Inanother embodiment, the method is as described above, wherein the IL-19polypeptide is administered in conjunction with a chemotherapeuticagent. In another embodiment, the method is as described above, whereinthe chemotherapeutic agent is selected from the group consisting ofpaclitaxel, cisplatin, carboplatin, topotecan, hexamethylmelamine,ifosfamide, doxorubicin, bleomycin, Taxol, and etoposide. In anotherembodiment, the method is as described above, wherein the IL-19polypeptide is fused with a cytotoxic moiety. In another embodiment, themethod is as described above, wherein the cytotoxic moiety is abacterial or plant toxin, cytotoxic radionuclide or cytotoxic drug.

[0049] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLE

[0050] We tested IL-19 in an Ovcar3 (ATCC #HTB-161) cytoxicity assay tomeasure the ability of IL-19 to prevent cells from growing during normalgrowth conditions. We used MTT reagent (Promega, Madison, USA) as ourdetection and readout for this cell inhibition assay. Procedure of acytoxicity assay: Ovcar3 Cytotoxicity Assay

[0051] Ovcar3 (ATCC #HTB-161) cells were plated at a density of 5000cells/100 ul/well in clear 96 well TC plates. Cells were plated incomplete growth media consisting of RPMI containing 20% FBS, 0.01 mg/mlinsulin, 2% HEPES, 1% Sodium Pyruvate and 1% Glutamax. Cells wereincubated overnight at 37° C. in a 5% CO2 incubator.

[0052] The following day, media was removed from the cells and replacedwith 100 ul/well of appropriately diluted samples. All sample dilutionswere done in complete growth media. Samples were incubated on the cellsfor 72 hours.

[0053] After incubation, an MTT assay was done on the cells using themanufacturer's protocol (Promega #PAG4100). Dye solution was incubatedon the cells 4 hours, followed by a 1 hour incubation with the stopsolution. Absorbance was then read on the Victor II and percentinhibition was calculated from the wells containing complete growthmedia only.

[0054] Results:

[0055] Retnoic Acid gave a 29% inhibition of growth at 3 uM, 34% at 10uM, 43% at 31 uM, and 83% at 100 uM (positive control)

[0056] IL-19 gave a 4% inhibition of growth at 1 ng/ml, 9% at 10 ng/ml,23% at 100 ng/ml and 52% at 1000 ng/ml.

[0057] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 2 1 985 DNA Homo sapiens CDS (63)...(593) 1 gaattcggca cgaggactgagaggagacac aaggagcagc ccgcaagcac caagtgagag 60 gc atg aag tta cag tgtgtt tcc ctt tgg ctc ctg ggt aca ata ctg 107 Met Lys Leu Gln Cys Val SerLeu Trp Leu Leu Gly Thr Ile Leu 1 5 10 15 ata ttg tgc tca gta gac aaccac ggt ctc agg aga tgt ctg att tcc 155 Ile Leu Cys Ser Val Asp Asn HisGly Leu Arg Arg Cys Leu Ile Ser 20 25 30 aca gac atg cac cat ata gaa gagagt ttc caa gaa atc aaa aga gcc 203 Thr Asp Met His His Ile Glu Glu SerPhe Gln Glu Ile Lys Arg Ala 35 40 45 atc caa gct aag gac acc ttc cca aatgtc act atc ctg tcc aca ttg 251 Ile Gln Ala Lys Asp Thr Phe Pro Asn ValThr Ile Leu Ser Thr Leu 50 55 60 gag act ctg cag atc att aag ccc tta gatgtg tgc tgc gtg acc aag 299 Glu Thr Leu Gln Ile Ile Lys Pro Leu Asp ValCys Cys Val Thr Lys 65 70 75 aac ctc ctg gcg ttc tac gtg gac agg gtg ttcaag gat cat cag gag 347 Asn Leu Leu Ala Phe Tyr Val Asp Arg Val Phe LysAsp His Gln Glu 80 85 90 95 cca aac ccc aaa atc ttg aga aaa atc agc agcatt gcc aac tct ttc 395 Pro Asn Pro Lys Ile Leu Arg Lys Ile Ser Ser IleAla Asn Ser Phe 100 105 110 ctc tac atg cag aaa act ctg cgg caa tgt caggaa cag agg cag tgt 443 Leu Tyr Met Gln Lys Thr Leu Arg Gln Cys Gln GluGln Arg Gln Cys 115 120 125 cac tgc agg cag gaa gcc acc aat gcc acc agagtc atc cat gac aac 491 His Cys Arg Gln Glu Ala Thr Asn Ala Thr Arg ValIle His Asp Asn 130 135 140 tat gat cag ctg gag gtc cac gct gct gcc attaaa tcc ctg gga gag 539 Tyr Asp Gln Leu Glu Val His Ala Ala Ala Ile LysSer Leu Gly Glu 145 150 155 ctc gac gtc ttt cta gcc tgg att aat aag aatcat gaa gta atg tcc 587 Leu Asp Val Phe Leu Ala Trp Ile Asn Lys Asn HisGlu Val Met Ser 160 165 170 175 tca gct tgatgacaag gaacctgtat agtgatccagggatgaacac cccctgtgcg 643 Ser Ala gtttactgtg ggagacagcc caccttgaaggggaaggaga tggggaaggc cccttgcagc 703 tgaaagtccc actggctggc ctcaggctgtcttattccgc ttgaaaatag ccaaaaagtc 763 tactgtggta tttgtaataa actctatctgctgaaagggc ctgcaggcca tcctgggagt 823 aaagggctgc cttcccatct aatttattgtgaagtcatat agtccatgtc tgtgatgtga 883 gccaagtgat atcctgtagt acacattgtactgagtggtt tttctgaata aattccatat 943 tttacctatg aaaaaaaaaa aaaaaaaagcggccgcctcg ag 985 2 177 PRT Homo sapiens 2 Met Lys Leu Gln Cys Val SerLeu Trp Leu Leu Gly Thr Ile Leu Ile 1 5 10 15 Leu Cys Ser Val Asp AsnHis Gly Leu Arg Arg Cys Leu Ile Ser Thr 20 25 30 Asp Met His His Ile GluGlu Ser Phe Gln Glu Ile Lys Arg Ala Ile 35 40 45 Gln Ala Lys Asp Thr PhePro Asn Val Thr Ile Leu Ser Thr Leu Glu 50 55 60 Thr Leu Gln Ile Ile LysPro Leu Asp Val Cys Cys Val Thr Lys Asn 65 70 75 80 Leu Leu Ala Phe TyrVal Asp Arg Val Phe Lys Asp His Gln Glu Pro 85 90 95 Asn Pro Lys Ile LeuArg Lys Ile Ser Ser Ile Ala Asn Ser Phe Leu 100 105 110 Tyr Met Gln LysThr Leu Arg Gln Cys Gln Glu Gln Arg Gln Cys His 115 120 125 Cys Arg GlnGlu Ala Thr Asn Ala Thr Arg Val Ile His Asp Asn Tyr 130 135 140 Asp GlnLeu Glu Val His Ala Ala Ala Ile Lys Ser Leu Gly Glu Leu 145 150 155 160Asp Val Phe Leu Ala Trp Ile Asn Lys Asn His Glu Val Met Ser Ser 165 170175 Ala

What is claimed is:
 1. A method for inhibiting the growth and orproliferation of ovarian cancer cells comprising bringing IL-19polypeptide into contact with the ovarian cancer cells in an amountsufficient to inhibit or reduce the proliferation of the ovarian cancercells.
 2. A method for treating a female mammal afflicted with ovariancancer comprising administering to the female an isolated IL-19polypeptide an amount of a composition of IL-19 polypeptide sufficientto inhibit or reduce the proliferation of the ovarian cancer.
 3. Themethod of claim 2, wherein the IL-19 polypeptide is administered inconjunction with radiation.
 4. The method of claim 2, wherein the IL-19polypeptide is administered in conjunction with a chemotherapeuticagent.
 5. The method of claim 4, wherein the chemotherapeutic agent isselected from the group consisting of paclitaxel, cisplatin,carboplatin, topotecan, hexamethylmelamine, ifosfamide, doxorubicin,bleomycin, Taxol, and etoposide.
 6. A method for treating a femalemammal afflicted with ovarian cancer comprising administering to thefemale an isolated IL-19 polypeptide an amount of a composition of IL-19polypeptide sufficient to inhibit or reduce the proliferation of theovarian cancer, and wherein the IL-19 polypeptide is fused with acytotoxic moiety.
 7. The method of claim 6, wherein the cytotoxic moietyis a bacterial or plant toxin, cytotoxic radionuclide or cytotoxic drug.8. A method of reducing proliferation of ovarian cancer cells comprisingadministering to a mammal with a ovarian neoplasm an amount of acomposition of IL-19 polypeptide sufficient to reduce proliferation ofthe neoplastic ovarian cells.
 9. The method of claim 8, wherein theIL-19 polypeptide is administered in conjunction with radiation.
 10. Themethod of claim 8, wherein the IL-19 polypeptide is administered inconjunction with a chemotherapeutic agent.
 11. The method of claim 9,wherein the chemotherapeutic agent is selected from the group consistingof paclitaxel, cisplatin, carboplatin, topotecan, hexamethylmelamine,ifosfamide, doxorubicin, bleomycin, Taxol, and etoposide.
 12. The methodof claim 8, wherein the IL-19 polypeptide is fused with a cytotoxicmoiety.
 13. The method of claim 12, wherein the cytotoxic moiety is abacterial or plant toxin, cytotoxic radionuclide or cytotoxic drug.